Hi,

I want to validate the results of my differentially expressed genes from RNA-Seq using qPCR (SYBR green). In my DESeq2, I used to consider covariates like Sex, Age, and cell type proportion and try to correct for them.

In my qPCR, I have the primers for Gene1, GFAP (astrocytes ), and MAP2 (Neurons), and two housekeeping genes.

Usually, I know to use the delta delta Ct method, whereby the Ct values in each sample are normalized to:

1) a housekeeper gene (or genes) within the same sample

2) the respective values in a control DNA sample

Like what Kevin Blighe explained here: How to report and plot qPCR data - delta delta Ct

But I don't know how to normalize my qPCR data for Gene1 for the GFAP (astrocytes ) and MAP2 (Neurons) as well?

Thank you very much in advance!